Preparation method and application of exosome of targeted macrophages
By regulating the glycosylation and surface modification of stem cells, exosomes targeting macrophages are prepared, which solves the problem that exosomes in existing technologies cannot target macrophages. It achieves precise targeting and efficient polarization of macrophages, effectively alleviating bone destruction in inflammatory diseases.
Patent Information
- Application Number
- CN202510773827.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-10-21
AI Technical Summary
In the existing technology, ordinary stem cell exosomes cannot target macrophages and the function of polarized macrophages is poor. There is a lack of engineered exosome preparation strategies targeting macrophages, and it is impossible to effectively regulate inflammatory bone dissolution.
By using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) and dibenzocyclooctyne-dextran sulfate (DBCO-DS) to regulate glycosylation and surface modification of stem cells, macrophage-targeted exosomes were prepared, and macrophage-targeted exosomes were extracted using serum-free culture medium and gradient ultrahigh-speed centrifugation.
The precise targeting and efficient polarization of exosomes to macrophages are achieved, which can effectively inhibit inflammatory bone dissolution and alleviate pathological bone destruction in inflammatory diseases such as rheumatoid arthritis, periprosthetic inflammation and periodontitis.
Smart Images
Figure CN120818484A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing exosomes targeting macrophages and applications thereof. Background Art
[0002] Macrophages, as core components of the innate immune system, play a complex and dynamic, bidirectional regulatory role in the pathophysiology of inflammatory diseases, such as osteoarthritis, spinal cord injury, and intervertebral disc degeneration. Macrophages exhibit remarkable plasticity, adopting two functional phenotypes in response to disease: classically activated (M1) and alternatively activated (M2). Classically activated (M1) macrophages are proinflammatory cells that can be polarized by lipopolysaccharide (LPS) or Th1 cytokines (such as IFN-γ and GM-CSF) to secrete proinflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-12, IL-23, and TNF-α. Alternatively activated (M2) macrophages possess anti-inflammatory and immunomodulatory properties and can be polarized by Th2 cytokines IL-4 and IL-13 to produce anti-inflammatory cytokines such as IL-10 and TGF-β. After bone and joint surgery, aseptic loosening caused by inflammatory osteolysis around the prosthesis is the main cause of prosthesis failure and the need for revision surgery. Under specific pathological conditions, M1 macrophages mediate osteoclast formation and inflammatory bone resorption through multiple biological effects.
[0003] Modulating the polarization of macrophages from M1 to M2 phenotype to reshape the inflammatory microenvironment in the area of osteolysis has become a research hotspot in recent years. Inducing macrophage polarization to the M2 phenotype effectively inhibits osteoclast maturation and activation. Therefore, targeted macrophage reprogramming to the M2 phenotype may provide a novel therapeutic strategy for inflammatory osteolysis.
[0004] Exosomes (Exos) are nanoscale vesicles secreted by cells, ranging in diameter from 40 to 160 nm. They contain bioactive substances such as cytokines, miRNAs, and long noncoding RNAs, similar to those of their source cells. Exosomes play a crucial role in intercellular communication, delivering bioactive substances and information from their source cells to effector cells. Exosomes derived from mesenchymal stem cells, in particular, have been extensively studied in the field of tissue repair and regeneration. Due to their low immunogenicity and high stability, exosomes (Exos) have recently demonstrated potential as key components of cell-free therapies for regulating inflammation and promoting bone regeneration. Studies have shown that cell-specific delivery of exosome contents is crucial for their therapeutic efficacy. However, strategies for exosome surface modification that target macrophages and enhance polarization efficiency are currently lacking.
[0005] The state of stem cells determines the biological function of the exosomes they are derived from. While stem cells have the potential for multidirectional differentiation (osteogenesis, chondrogenesis, and adipogenesis), conventional stem cell exosomes lack the ability to target macrophages. This limits the biological function of umbilical cord stem cell exosomes in treating inflammatory osteolysis by inhibiting inflammatory responses. Currently, there are no studies or reports on the use of engineered umbilical cord stem cell exosomes targeting and polarizing macrophages to treat inflammatory osteolysis. Summary of the Invention
[0006] The present invention is directed to the above-mentioned problems and provides macrophage-targeted extracellular vesicles derived from umbilical cord mesenchymal stem cells (MT-Exos) and a preparation method and application thereof, so as to solve the technical problems in the prior art that ordinary stem cell exosomes cannot target macrophages and have poor function in polarizing macrophages.
[0007] The research strategy of this invention is as follows: stem cells are first stimulated with N-azidoacetylmannosamine tetraacylated (Ac4ManNAz) to regulate their glycosylation and improve their epigenetic state. Dibenzocyclooctyne-dextran sulfate (DBCO-DS) is then used to stimulate and modify the pretreated stem cells. After culturing the stem cells, macrophage-targeted stem cell exosomes are obtained. These exosomes have been shown to effectively regulate the transition of macrophage polarization phenotype from M1 to M2.
[0008] Based on the above research, the technical solutions to be protected by the present invention are as follows:
[0009] In a first aspect, the present invention provides a method for preparing exosomes targeting macrophages, using stem cells as raw materials, comprising the following steps: (1) stimulating stem cells with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) to a preset cell fusion degree; (2) subsequently adding dibenzocyclooctyne-dextran sulfate (DBCO-DS) to stimulate the above-pretreated stem cells and perform surface modification on the stem cells; (3) culturing the stem cells obtained in step (2) in a serum-free medium, collecting the cell culture supernatant, and obtaining stem cell exosomes targeting macrophages by gradient centrifugation and ultrahigh-speed centrifugation.
[0010] The preferred process conditions for the above steps are as follows:
[0011] In step (1), the stem cells are selected from mesenchymal stem cells and pluripotent stem cells, such as bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or embryonic stem cells;
[0012] The concentration of Ac4ManNAz is 10-40 μM, and the stimulation time is ≥24 hours; the preset cell confluence is ≥85%, preferably 85%, 90%, and 95%.
[0013] In the specific embodiments of the present invention, Ac4ManNAz was used to stimulate umbilical cord mesenchymal stem cells at a gradient concentration of 5-40 μM. The results showed that only when the concentration range was 10-40 μM, it had the effect of promoting stem cell transformation.
[0014] In step (2), the stimulation concentration of DBCO-DS is 10-50 μM, and the stimulation time is 1-6 hours.
[0015] In step (3), serum-free medium (SFM) is obtained from existing technology. It has clear ingredients, high safety, and can control directional differentiation. It has important applications in stem cell culture. It can avoid batch differences, pathogen risks, and interference from unknown components caused by animal serum (such as FBS), and is more conducive to standardized culture and clinical application.
[0016] The centrifugal forces involved in gradient centrifugation are 300×g, 1200×g, and 2500×g, respectively. The centrifugation condition is 4°C, and the gradient centrifugation time is 15-30 minutes, for example, 15 minutes, 20 minutes, 25 minutes, and 30 minutes. Gradient centrifugation can separate cell debris and apoptotic cells, improving the efficiency of exosome purification.
[0017] The ultra-high-speed centrifugation method has a centrifugal force of ≥100,000×g, centrifugation conditions: 4° C., time ≥70 minutes, preferably 90 minutes.
[0018] In the present invention, a filtration step is preferably added between the gradient centrifugation and ultracentrifugation extraction steps. Residual cell debris can be removed by filtering the supernatant after the gradient centrifugation. The filter size is 0.22 μm.
[0019] In a second aspect, the present invention provides macrophage-targeted exosomes prepared using the above method. The exosomes are approximately elliptical in shape, with a particle size concentrated around 126.7 nm, and can detect exosomal markers CD63, CD9, CD81, and EFA1.
[0020] In a third aspect, the present invention provides applications of the aforementioned macrophage-targeted exosomes. The first application is in the preparation of a formulation for regulating the conversion of macrophage polarization phenotype from M1 to M2; the second application is in the preparation of a drug for treating inflammatory osteolysis; and the third application is in the preparation of a drug for treating inflammatory diseases.
[0021] Experiments have confirmed that the exosomes prepared by the method of the present invention can effectively achieve the conversion of macrophage polarization phenotype from M1 to M2; inducing macrophage polarization to M2 phenotype can effectively inhibit the maturation and activation of osteoclasts, and can be used to alleviate inflammatory osteolysis (referring to the pathological bone destruction process caused by chronic inflammation (such as rheumatoid arthritis, periprosthetic inflammation, periodontitis or tumor-related inflammation)), and further has an alleviating effect on inflammatory diseases involving classically activated (M1) macrophages.
[0022] The beneficial protection and effects of the present invention are as follows:
[0023] The macrophage-targeted umbilical cord mesenchymal stem cell exosomes extracted using the technical methods provided herein specifically bind to the scavenger receptor type A (SR-A) on the surface of macrophages through surface-modified dextran sulfate (DS), achieving cell-specific delivery. SR-A is a membrane protein unique to macrophages, particularly highly expressed during inflammatory activation. This allows MT-Exos to precisely target macrophages in lesion areas, avoiding nonspecific differentiation. Conventional exosomes, on the other hand, lack active targeting and rely on passive uptake, resulting in lower efficiency. Furthermore, the ligand-receptor binding of DS to SR-A not only improves uptake efficiency but also regulates macrophage polarization. Targeted binding of MT-Exos to macrophages can directly suppress pro-inflammatory phenotypes (such as M1) and induce polarization toward an anti-inflammatory phenotype (such as M2), thereby more effectively suppressing the inflammatory microenvironment and inhibiting inflammatory diseases. Therefore, the present invention provides a new approach for the treatment of inflammatory diseases involving macrophages. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 This is a transmission electron microscopy image of the exosomes MT-Exos extracted in Example 1.
[0025] Figure 2 This is a picture of the particle size analysis of exosomes MT-Exos extracted in Example 1.
[0026] Figure 3 This is a Western blot image of the exosome MT-Exos extracted in Example 1.
[0027] Figure 4 This is an EFA1 immunofluorescence image of MT-Exos secreted by stem cell exosomes carrying the DBCO-DS label in Example 2, where EFA1 is a marker of exosomes in the early stage of cell exosomes.
[0028] Figure 5This is a CD63 immunofluorescence image of MT-Exos secreted by stem cell exosomes carrying the DBCO-DS label in Example 2, where CD63 is a marker of exosomes.
[0029] Figure 6 These are the immunofluorescence images and statistical analysis of the exosomes MT-Exos extracted in Example 1 regulating M1 macrophages, where iNOS is a marker for M1 macrophages.
[0030] Figure 7 These are the immunofluorescence images and statistical analysis of the exosomes MT-Exos extracted in Example 1 regulating M2 macrophages. CD206 is a marker for M2 macrophages. DETAILED DESCRIPTION
[0031] The present invention will now be described in detail with reference to the embodiments and the accompanying drawings, but the implementation of the present invention is not limited thereto. The technical solutions in the embodiments of the present application will be clearly and completely described below. However, the embodiments described are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work fall within the scope of protection of the present application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased on the market or can be prepared by existing methods.
[0032] Macrophages possess remarkable plasticity, polarizing into two phenotypes in response to disease: classically activated (M1) and alternatively activated (M2). M1 macrophages are closely associated with proinflammatory responses and elevated levels of reactive oxygen species. Regulating macrophage polarization to convert the M1 phenotype to the M2 phenotype can effectively suppress the inflammatory microenvironment.
[0033] Exosomes (Exos), due to their low immunogenicity and high stability, have recently shown potential as key components of cell-free therapies for regulating inflammation and promoting bone regeneration. Research has shown that cell-specific delivery of exosome contents is crucial for achieving therapeutic efficacy, but strategies for exosome surface modification that target macrophages and enhance polarization efficiency are currently lacking.
[0034] Given the importance of suppressing the inflammatory microenvironment for the treatment of inflammatory diseases, how to construct exosomes that target macrophages and enhance macrophage polarization efficiency to meet the treatment needs of inflammatory diseases is a scientific problem that needs to be solved urgently. The specific technical solution is as follows:
[0035] Example 1 Preparation of exosomes targeting macrophages
[0036] A. Ac4ManNAz stimulates stem cells
[0037] (1) Umbilical cord mesenchymal stem cells were stimulated with 5-40 μM Ac4ManNAz, and the stimulation time was set to 24 hours. 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, and 40 μM were marked as AH groups, respectively.
[0038] (2) The cells were then fixed with 4% paraformaldehyde and the nuclei were stained with 2 μg / mL DAPI. The attachment of azide groups on the surface of umbilical cord mesenchymal stem cells in each group was observed under a confocal microscope under the stimulation of various concentrations of Ac4ManNAz. Based on the above results, the appropriate concentration of Ac4ManNAz was selected to stimulate umbilical cord mesenchymal stem cells. The results showed that 10-40 μM Ac4ManNAz was effective.
[0039] B. DBCO-DS stimulation
[0040] After treatment in step A, Cy5.5-labeled dibenzocyclooctyne-dextran sulfate (DBCO-DS) was added to the stem cells to stimulate the pretreated stem cells. The DBCO-DS stimulation concentration ranged from 10 to 50 μM, and the stimulation duration ranged from 1 to 6 hours. 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM concentrations were labeled as groups A1 to H1, respectively, and the stimulation duration was 1 hour. Repeat the above concentrations for a stimulation duration of 3 hours, labeled as groups A3 to H3. Repeat the above concentrations for a stimulation duration of 6 hours, labeled as groups A6 to H6. Exosomes from engineered umbilical cord mesenchymal stem cells were screened for exosomes that responded to the stimulation concentration and duration using EFA1 immunofluorescence staining (see Example 2 for detailed fluorescence staining methods).
[0041] C. Exosome extraction
[0042] (1) When the engineered umbilical cord mesenchymal stem cells are cultured to a predetermined cell confluence, the medium is changed to serum-free medium and cultured for 48 hours. The predetermined cell confluence is ≥85%, which can be 85%, 90%, or 95%.
[0043] (2) Collect the supernatant of the cell culture medium and separate the cell debris and apoptotic cells by gradient centrifugation. The centrifugal forces involved in the gradient centrifugation are 300×g, 1200×g, and 2500×g, respectively, and each centrifugation is 15 minutes. Gradient centrifugation can separate cell debris and apoptotic cells, thereby improving the purification efficiency of exosomes.
[0044] (3) Filter the supernatant after centrifugation using a 0.22 μm filter. This method can filter out residual cell debris.
[0045] (4) Exosomes were extracted using ultrahigh-speed centrifugation at a centrifugal force of 100,000 × g for 90 min at 4°C.
[0046] (5) Resuspend the exosomes in phosphate solution pre-cooled at 4°C to obtain engineered umbilical cord mesenchymal stem cell exosomes (MT-Exos) that can target macrophages and store them at -80°C.
[0047] The exosomes obtained were detected and found to be approximately oval in shape ( Figure 1 ), the particle size is concentrated around 126.7nm ( Figure 2 ), and exosome markers CD63, CD9 and CD81 were detected ( Figure 3 ).
[0048] Example 2 Exosome fluorescence detection
[0049] Based on the preparation method disclosed in Example 1, the engineered umbilical cord mesenchymal stem cell exosomes obtained under the conditions of adapted stimulation concentration and time were screened by immunofluorescence staining of EFA1.
[0050] Cells in groups A1-H1, A2-H2, and A3-H3 were fixed with 4% paraformaldehyde for 10 minutes, washed twice with PBS for 5 minutes each time, and then permeabilized with 0.25% Triton-X100 for 10 minutes. The cells were blocked with 200 μL of 5% goat serum at room temperature for 1 hour. The rabbit anti-EFA1 primary antibody dilution was prepared at a ratio of 1:500 using PBS as the solvent, and 200 μL of the primary antibody dilution was added to each well, and the cells were incubated at 4°C for 12 hours.
[0051] The primary antibody was recovered and the cells were washed twice with PBS for 5 minutes each time. Then, 200 μL of FITC goat anti-rabbit secondary antibody was added at a dilution ratio of 1:500.
[0052] The secondary antibody was removed by aspiration, and each well was washed twice with PBS for 5 minutes each time, followed by addition of 200 μL DAPI staining solution and incubation at room temperature for 10 minutes.
[0053] The cell slides were removed and placed on a glass slide. Anti-fading mounting medium was added and the slides were sealed. The expression of EFA1 in different groups was observed under a confocal electron microscope with an excitation wavelength of 488 nm. Figure 3 and Figure 4 As shown, EFA1 and CD63 markers were expressed.
[0054] Example 3 Exosomes can promote macrophage polarization
[0055] Based on the preparation method disclosed in Example 1, further verification that the exosomes can promote the polarization of macrophages was performed, which specifically included the following steps:
[0056] (1) RAW264.7 cells were seeded into confocal microplates at a cell confluence of 30%;
[0057] (2) Culture in RPMI1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 500 ng / mL lipopolysaccharide (LPS) for 24 hours;
[0058] (3) Add the above-mentioned engineered umbilical cord mesenchymal stem cell exosomes (MT-Exos) and ordinary umbilical cord mesenchymal stem cell exosomes (Exos) and co-culture for 48 hours;
[0059] (4) Add 4% paraformaldehyde to fix the cells for 10 minutes;
[0060] (5) Wash twice with PBS for 5 minutes each time, and then permeabilize with 0.25% Triton-X100 for 10 minutes;
[0061] (6) Block with 200 μL of 5% goat serum at room temperature for 1 hour;
[0062] (7) Prepare rabbit anti-iNOS and rabbit anti-CD206 primary antibody diluents at a ratio of 1:500, using PBS as the solvent. Add 200 μL of primary antibody diluent to each well and incubate at 4°C for 12 hours.
[0063] (8) Recover the primary antibody and wash the cells twice with PBS for 5 minutes each time. Then add 200 μL of FITC goat anti-rabbit secondary antibody at a dilution ratio of 1:500.
[0064] (9) Remove the secondary antibody by aspiration, add PBS to each well and wash twice, 5 minutes each time, then add 200 μL DAPI staining solution and incubate at room temperature for 10 minutes.
[0065] (10) Take out the cell slide and place it on a glass slide. Add anti-quenching mounting medium and seal the slide.
[0066] (11) The expression of iNOS and CD206 in different groups was observed under confocal electron microscopy. The excitation wavelength was 488 nm.
[0067] The number of cells expressing iNOS and CD206 was counted. Figure 6 As shown in the figure, it is suggested that the exosomes prepared by the present invention can regulate the conversion of macrophage polarization phenotype from M1 to M2.
[0068] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing exosomes targeting macrophages, characterized in that: The steps include: (1) Stimulating stem cells with N-azidoacetylmannosamine-tetraacylated Ac4ManNAz to a predetermined cell confluency; (2) Subsequently, dibenzocyclooctyne-dextran sulfate DBCO-DS was added to stimulate the pretreated stem cells to modify the stem cell surface; (3) After culturing the stem cells obtained in step (2) in a serum-free medium, the cell culture supernatant is collected and macrophage-targeted stem cell exosomes are obtained by gradient centrifugation and ultrahigh-speed centrifugation.
2. The preparation method according to claim 1, characterized in that In step (1), the stem cells are selected from mesenchymal stem cells and pluripotent stem cells, and the pluripotent stem cells are selected from bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells or embryonic stem cells.
3. The preparation method according to claim 1, characterized in that In step (1), the concentration of Ac4ManNAz is 10-40 μM, the stimulation time is ≥24 hours, and the preset cell confluence is ≥85%.
4. The preparation method according to claim 1, characterized in that In step (2), the stimulation concentration of DBCO-DS is 10-50 μM, and the stimulation time is 1-6 hours.
5. The preparation method according to claim 1, characterized in that In step (3), the culture time in serum-free medium is 2 days; The centrifugal forces involved in the gradient centrifugation method are 300×g, 1200×g, and 2500×g, respectively, and the gradient centrifugation time is 15-30 minutes; The centrifugal force of the ultra-high-speed centrifugation method is ≥100,000×g, the centrifugation conditions are: 4°C, and the time is ≥70 minutes.
6. The preparation method according to claim 1, characterized in that In step (3), the supernatant obtained by gradient centrifugation is filtered using a 0.22 μm filter before ultracentrifugation.
7. An exosome targeting macrophages, characterized in that The method according to any one of claims 1 to 6 is used for preparation.
8. Use of the exosomes according to claim 7 in preparing a preparation for regulating the conversion of macrophage polarization phenotype from M1 to M2.
9. Use of the exosomes according to claim 7 in the preparation of a drug for treating inflammatory osteolysis diseases.
10. Use of the exosomes according to claim 7 in preparing a drug for treating inflammatory diseases.